Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, presents clinical challenges. Unlike adrenergic (ADRN) neuroblastoma cells, neuroblastoma cells with a mesenchymal (MES) identity are resistant to chemotherapy and retinoid therapy, which contributes to relapses and treatment failures. We explored whether up-regulation of the neurogenic, tumor suppressor microRNA miR-124 could promote the differentiation of retinoic acid-resistant MES neuroblastoma cells. Leveraging our screen for miRNA-modulatory small molecules, we identified and validated the tyrosine and phosphoinositide kinase inhibitor PP121 as a robust inducer of miR-124. Combining PP121 and BDNF-activating bufalin synergistically arrested proliferation and promoted the sustained differentiation of MES/heterogeneous SK-N-AS cells over several weeks. This protocol also resulted in the differentiation of multiple MES neuroblastoma and glioblastoma cell lines. RNA-seq analysis of differentiated MES/heterogeneous SK-N-AS cells revealed the replacement of the ADRN core regulatory circuitry with circuitries associated with chromaffin cells and Schwann cell precursors. Furthermore, differentiation was associated with inhibition of the CDK4/CDK6 pathway and activation of a transcriptional program that correlated with improved outcomes for patients with neuroblastoma. Our findings suggest an approach with translational potential to induce the differentiation of therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells could be used to study mechanisms underlying cancer biology and therapies.